During the course of infection, bacterial pathogens are dependent upon the secretion of multiple protein products that modulate host cell physiology and facilitate bacterial growth. A number of protein secretion systems have been identified and functionally characterized for gram-negative bacteria for which the existence of both an inner and outer membrane presents a significant barrier to protein translocation (Cascales (2008) EMBO Rep. 9:735-41; Desvaux, et al. (2009) Trends Microbiol. 17:139-145; Donnenberg (2000) Nature 406:768-74; Gerlach & Hensel (2007) Int. J. Med. Microbiol. 297:401-15; Marlovits (2009) Curr. Opin. Microbiol. 13:47-52; Russel (1998) J. Mol. Biol. 279:485-99; Young, et al. (1999) Proc. Natl. Acad. Sci. USA 96:6456-61). In gram-positive bacteria, secreted proteins are translocated across the single bacterial cell membrane in an unfolded state and delivered to the compartment existing between the membrane and the cell wall (Sarvas, et al. (2004) Biochim. Biophys. Acta 1694:311-27). The cell walls of gram-positive bacteria are composed of a thick matrix of peptidoglycan layers and glycopolymers, including teichoic acids and lipoteichoic acids (Weidenmaier & Peschel (2008) Nat. Rev. Microbiol. 6:276-87), and these abundant anionic polymers have a high capacity to bind divalent metal ions and cationic molecules (Beveridge & Murray (1980) J. Bacteriol. 141:876-887; Wahlstrom, et al. (2003) Microbiology 149:569-77). Proteins that are translocated across the bacterial membrane therefore enter a challenging environment for protein folding based on the high density of negative charge, high concentrations of cations, and low pH (Sarvas, et al. (2004) Biochim. Biophys. Acta 1694:311-27; Wahlstrom, et al. (2003) Microbiology 149:569-77). Within this environment, secreted proteins may additionally require further posttranslational modification, proteolytic activation, or sequestration prior to release for interaction with host cell targets. It should be noted that not all secreted proteins are found in the extracellular milieu, as many are specifically localized at the membrane or within the cell wall. Proteins present in bacterial culture supernatants thus constitute a group of exoproteins to which numerous pathogenic traits can be attributed (Desvaux, et al. (2009) Trends Microbiol. 17:139-145).
For the facultative intracellular pathogen Listeria monocytogenes, protein secretion has been reported to occur primarily via the Sec-mediated secretion pathway (Desvaux & Hebraud (2006) FEMS Microbiol. Rev. 30:774-805). Proteins secreted via Sec-dependent secretion include well-characterized virulence factors, such as the internalins InlA and InlB, which mediate host cell invasion (Bierne, et al. (2007) Microbes Infect. 9:1156-66; Gaillard, et al. (1991) Cell 65:1127-41; Ireton (2007) Cell Microbiol. 9:1365-75; Kim, et al. (2004) Infect. Immun. 72:7374-78; Kim et al. (2005) Microbiology 151:3215-222; Lingnau, et al. (1995) Infect. Immun. 63:3896-903; Pizarro-Cerda & Cossart (2006) J. Pathol. 208:215-223; Seveau, et al. (2007) Microbes Infect. 9:1167-75), listeriolysin-O (LLO) and the broad-range phosphatidyl-choline phospholipase (PC-PLC), which mediate vacuole membrane lysis (Dramsi & Cossart (2003) Infect. Immun. 71:3614-8; Glomski, et al. (2003) Infect. Immun. 63:3896-903; Glomski, et al. (2002) J. Cell Biol. 156:1029-38; Grundling, et al. (2003) J. Bacteriol. 185:6295-307; Henry, et al. (2006) Cell. Microbiol. 8:107-19; Marquis & Hager (2000) Mol. Microbiol. 35:289-98; Schnupf, et al. (2006) Mol. Microbiol. 61:999-1012; Schnupf & Portnoy (2007) Microbes Infect. 9:1176-87; Schnupf, et al. (2007) Infect. Immun. 75:5135-47; Vazquez-Boland, et al. (1992) Infect. Immun. 60:219-230; Yeung, et al. (2007) Infect. Immun. 75:44-51), and the surface protein ActA, which mediates actin polymerization and cell-to-cell spread within the host (Auerbuch, et al. (2001) J. Biol. Chem. 281:31812-22; Boujemaa-Paterski, et al. (2001) Biochemistry 40:11390-404; Domann, et al. (1992) EMBO J. 11:1981-90; Kocks, et al. (1992) Cell 68:521-31; Skoble, et al. (2001) J. Cell Biol. 155:89-100; Skoble, et al. (2000) J. Cell Biol. 150:527-38; Smith & Portnoy (1997) Trends Microbiol. 5:272-6; Wong, et al. (2004) Cell Microbiol. 6:155-66). These proteins are critical for the establishment of the L. monocytogenes replication niche within the cytosol of infected host cells (Freitag (2006) Future Microbiol. 1:89-101; Gray, et al. (2006) Infect. Immun. 74:2505-12; Scortti, et al. (2007) Microbiol. Infect. 9:1196-1207; Vazquez-Boland, et al. (1990) Antimicrob. Agents Chemother. 34:539-42).
L. monocytogenes PrsA1 and PrsA2 are secreted proteins that are predicted to function as parvulin-type peptidyl-prolyl isomerase (PPIase) chaperones at the bacterial membrane-cell wall interface to assist in the folding and stability of secreted proteins (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23). PrsA2 appears to be primarily adapted for L. monocytogenes pathogenesis, based on the regulation of prsA2 expression by the central virulence transcriptional activator PrfA and on the essential requirement for PrsA2 for bacterial virulence in mice (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23; Port & Freitag (2007) Infect. Immun. 75:5886-97; Zemansky, et al. (2009) J. Bacteriol. 191:3950-64). The loss of PrsA2 dramatically reduces bacterial cell-to-cell spread in monolayers of mouse fibroblast cells and also reduces LLO stability and impedes the processing of PC-PLC to its enzymatically active form (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23; Chatterjee, et al. (2006) Infect. Immun. 74:1323-38; Zemansky, et al. (2009) J. Bacteriol. 191:3950-64). Further, it has been demonstrated that prsA2 deletion mutants are defective for bacterial flagellum-mediated swimming motility, an observation that suggests multiple roles for PrsA2 both inside and outside infected host cells (Zemansky, et al. (2009) J. Bacteriol. 191:3950-64). In contrast to its homologue in Bacillus subtilis, PrsA2 is not required for L. monocytogenes viability, and LprsA2 mutants replicate very similarly to wild-type strains in broth culture and on agar medium (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23; Sarvas, et al. (2004) Biochim. Biophys. Acta 1694:311-27).
Unlike L. monocytogenes LprsA2 mutants, strains lacking prsA1 are fully virulent in mouse models of infection (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23). prsA1 is not required for bacterial growth in broth culture, and its potential contributions to other aspects of L. monocytogenes physiology are as yet undefined. PrsA2 and PrsA1 are highly similar at the amino acid sequence level; thus, it is possible that PrsA2 and PrsA1 share some degree of functional overlap (Alonzo, III et al. (2009) Infect. Immun. 77:2612-23). In B. subtilis, the depletion of PrsA leads to the induction of the CssR/S two-component system and increased expression of the HtrA chaperone/protease in response to the accumulation of misfolded proteins at the bacterial membrane-cell wall interface (Hyyrylainen, et al. (2001) Mol. Microbiol. 41:1159-72; Sarvas, et al. (2004) Biochim. Biophys. Acta 1694:311-27). The loss or depletion of both PrsA2 and PrsA1 in L. monocytogenes could potentially elicit a similar membrane stress response if one or both are required for the folding of a large number of secreted proteins.
Initially characterized in Escherichia coli, HtrA is one of several proteins, collectively known as heat shock proteins, whose expression is essential for survival of bacteria at high temperatures. In addition, htrA has been shown to be essential for the pathogenicity of several gram-negative and gram-positive bacteria, namely, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, Streptococcus pyogenes, and Streptococcus pneumonia, as well as the antibiotic stress response in Lactococcus lactis and Staphylococcus aureus. Analysis of HtrA in listerial pathogenesis revealed a ˜1-log reduction in the level of the htrA mutant relative to the wild-type 3 days after intraperitoneal infection (Stack, et al. (2005) Appl. Environ. Microbiol. 71:4241-7). Likewise, the survival of a htrA prsA2 double mutant has been shown to be significantly impaired in mice (Alonzo, III & Freitag (2010) Infect. Immun. 70(11):4944-4257).
L. monocytogenes has been shown to exhibit the ability to induce an innate immune response that leads to robust and highly functional CD4 and CD8 T cell immunity specific for vaccine-encoded antigens. see, e.g., US 2003/0202985; US 2002/0136737; Angelakopoulos, et al. (2002) Infect. Immun. 70:3592-601; Johnson, et al. (2011) Microbiol. Immunol. 55:304-17; Mathew, et al. (2005) J. Immunol. 174:2212-9; and Radulovic, et al. (2009) J. Buon. 14:(suppl. 1):S165-8.